Influenza A viruses (IAV) are significant human pathogens causing yearly epidemics and occasional pandemics. Past pandemics have resulted in significant morbidity and mortality. The 1918 influenza pandemic was thought to have resulted in the death of at least 675,000 people in the U.S., and 40 million people worldwide. Pandemics in 1957 and 1968, while less severe, were also of major public health importance. A novel influenza A virus of swine origin became pandemic in 2009, causing the first pandemic in 41 years. In addition, annual epidemic influenza causes are also very significant resulting in approximately 36,000 deaths in the US annually. The influenza A virus hemagglutinin (HA) is responsible for binding sialic acid containing glycoproteins on cells to initiate infection. The specificity of HA binding to terminal sialic acid (SA) residues is dependent on the conformation of the bond to the penultimate carbohydrate. In general, avian-adapted IAVs have a binding preference for SA alpha2,3 Gal and human-adapted IAVs have a binding preference for SA alpha2,6 Gal. To understand how changing HA binding specificity affects IAV virulence in a mouse model, we studied the growth, virulence and pathogenicity of a number of recombinant influenza viruses expressing wild-type and mutant avian and human HA genes created using reverse genetics on the backbone of a contemporary low passage human H1N1 virus. Chimeric influenza viruses with the hemagglutinin segment of the 1918, 1957, 1968, or 2009 pandemic influenza viruses in the context of a seasonal H1N1 influenza genome were constructed to analyze the role of hemagglutinin (HA) in pathogenesis and cell tropism in a mouse model. We also explored whether there was an association between the ability of lung surfactant protein D (SP-D) to bind to the HA and the ability of the corresponding chimeric virus to infect bronchiolar and alveolar epithelial cells of the lower respiratory tract. Viruses expressing the hemagglutinin of pandemic viruses were associated with significant pathology in the lower respiratory tract, including acute inflammation, and showed low binding activity for SP-D. In contrast, the virus expressing the HA of a seasonal influenza strain induced only mild disease with little lung pathology in infected mice and exhibited strong in vitro binding to SP-D. In collaboration with Paul Digard, Cambridge University, the regulation of expression of different viral proteins encoded by the influenza A virus segment 2 (PB1) was examined. The PB1 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, AUGs 1-4 were systematically mutated and polypeptide synthesis was monitored from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. Overall, it was concluded that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging. Secondary bacterial infections increase disease severity of influenza infections and contribute greatly to increased morbidity and mortality during pandemics, including the 1918 and 2009 H1N1 pandemics. To study secondary bacterial infection following primary influenza virus infection, BALB/c mice were inoculated with sub-lethal doses of 2009 seasonal H1N1 virus (NIH50) or 2009 pandemic H1N1 virus (Mex09) followed by inoculation with Streptococcus pneumoniae (SP) 48h later. Disease course was characterized by weight loss, survival, titration of virus and bacteria by qRT-PCR and histopathology. Host responses were studied by expression microarray and immunohistochemistry. Mice inoculated with virus alone showed 100% survival for all groups. Mice inoculated with Mex09+SP showed severe weight loss and 100% mortality with severe alveolitis, denuded bronchiolar epithelium and widespread expression of apoptosis marker cleaved caspase 3. In contrast, mice inoculated with NIH50+SP showed increased weight loss, 100% survival and slightly enhanced lung pathology. Mex09+SP co-infection resulted in increased SP replication in lung, and bacteremia that occurred late in infection. Global gene expression profiling revealed that Mex09+SP co-infection did not induce significantly more severe inflammatory responses, but featured significant loss of epithelial cell reproliferation and repair responses. Histopathological examination for cell proliferation marker MCM7 showed significant staining of airway epithelial cells in all groups except Mex09+SP. This study demonstrated that secondary bacterial infection during 2009 H1N1 pandemic virus infection resulted in increased bacterial replication, more severe disease and loss of lung repair responses compared to 2009 seasonal influenza viral and bacterial infection. Moreover, this study provides novel insights into influenza and bacterial co-infection by showing a correlation of lethal outcome with loss of airway basal epithelial cells and associated lung repair responses.